Engineered Polynucleotides for RNA Base Editing

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Solution Overview

Problem

Current methods for modulating splicing activity and RNA base editing are inefficient, non-specific, and face challenges in delivery, limiting their effectiveness in clinical applications for diseases such as Rett syndrome, Huntington's disease, and muscular dystrophy.

Innovation Solution

Engineered polynucleotides are developed, incorporating a targeting sequence with mismatches and an Sm or Sm-like protein binding domain, along with a hairpin, to enhance specificity and efficiency of RNA base editing by directing endogenous ADAR deaminase activity to target genes, including those associated with specific diseases.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If conventional guide RNAs are used for RNA base editing and splicing modulation, then the approach can be implemented, but the efficiency, specificity, and delivery are insufficient for clinical applications

Engineering Contradiction:

Improveefficiency and specificity of RNA base editingVSAvoidclinical effectiveness

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent modifies the guide RNA structure by incorporating a U7 snRNA scaffold with specific structural elements (Sm binding domain, hairpin structure) and optimized parameters (length 30-300 bases, specific mismatch positions) to enhance stability, nuclear retention, and editing efficiency, resolving the contradiction between reliability and clinical effectiveness

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention creates a composite guide RNA structure combining multiple functional elements: U7 snRNA scaffold, Sm binding domain, hairpin structure, and targeting sequence with specific mismatches. This composite design integrates stability, specificity, and delivery capabilities into a single molecule, improving both reliability and clinical productivity

Inventive Principle:

Principle #40Composite materials

2Manufacturing precision

If the targeting sequence is made longer to improve specificity, then binding accuracy increases, but the RNA stability and delivery efficiency may decrease

Engineering Contradiction:

Improvespecificity of target bindingVSAvoidRNA stability and retention

Core Design Contradiction:

Manufacturing precisionVSStability of the object's composition

Solution Approach 1:

The patent optimizes the targeting sequence length parameter to 30-300 bases and positions mismatches at specific locations (45-55 bases from ends) to achieve the optimal balance between binding specificity and RNA stability, preventing both degradation and off-target effects

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention introduces localized mismatched nucleotides at specific positions within the targeting sequence rather than uniform composition throughout. This local modification enhances target recognition specificity while maintaining overall RNA structural integrity and stability

Inventive Principle:

Principle #3Local quality

3Manufacturing precision

If mismatches are introduced in the targeting sequence to improve editing specificity, then off-target effects are reduced, but the binding strength to target RNA may be weakened

Engineering Contradiction:

Improveediting specificityVSAvoidbinding strength

Core Design Contradiction:

Manufacturing precisionVSForce

Solution Approach 1:

The patent specifies that mismatches be positioned 45-55 bases from either end of the targeting sequence, optimizing their location to maximize editing specificity while minimizing impact on overall binding strength through strategic placement rather than random distribution

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The U7 snRNA scaffold acts as an intermediary structure that compensates for the binding strength reduction caused by mismatches. The scaffold provides additional stabilization through its structured domains (Sm binding domain, hairpin), allowing mismatches to be used for specificity without sacrificing necessary binding affinity

Inventive Principle:

Principle #24Intermediary (Mediator)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

The engineered polynucleotides significantly improve the efficiency and specificity of splicing activity modulation and RNA base editing, achieving up to 50% increased exon skipping efficiency and precise editing of target RNAs, as demonstrated by droplet digital PCR assays and Sanger sequencing.

Implementation Method 1

a targeting sequence that at least partially hybridizes to at least a portion of a target RNA

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

an Sm or Sm-like protein binding domain or variant thereof from a spliceosomal snRNA

Methodology Applied

Scientific EffectProtein-RNA binding:

Implementation Method 3

a hairpin from a spliceosomal snRNA or a non-spliceosomal snRNA or a variant thereof

Methodology Applied

Scientific EffectHairpin formation:

Implementation Method 4

directing endogenous ADAR deaminase activity to desired gene targets. This RNA base editing can occur within coding sequences

Methodology Applied

Scientific EffectDeamination:

Data Source

PatentUS20240011028A9Compositions and Methods Using SNRNA Components

Publication Date: 2024.01.11 SHAPE THERAPEUTICS INC

AI summary

Disclosed herein are compositions, pharmaceutical compositions, and methods of use comprising an engineered polynucleotide that can be used to hybridize with a target RNA which may contain a nucleotide mismatch. Compositions and methods disclosed herein can be used to edit RNA to ameliorate or treat diseases or conditions in a subject.